Method and apparatus for hybridization

ABSTRACT

A body  300  having a cavity  310  for mounting a substrate  120  fabricated with probe sequences at known locations according to the methods disclosed in U.S. Pat. No. 5,143,854 and PCT WO 92/10092 or others, is provided. The cavity includes inlets  350  and  360  for introducing selected fluids into the cavity to contact the probes. Accordingly, a commercially feasible device for use in high throughput assay systems is provided.

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent applicationSer. No. 09/302,052, filed Apr. 29, 1999, which is a continuation ofU.S. patent application Ser. No. 08/485,452, filed Jun. 7, 1995, nowU.S. Pat. No. 5,945,334, which is continuation-in-part U.S. patentapplication No. 08/255,682, filed Jun. 8, 1994. Each of theseapplications is incorporated herein by reference in its entirety for allpurposes.

BACKGROUND OF THE INVENTION

[0002] The present inventions relate to the fabrication and placement ofmaterials at known locations on a substrate. In particular, oneembodiment of the invention provides a method and associated apparatusfor packaging a substrate having diverse sequences at known locations onits surface.

[0003] Techniques for forming sequences on a substrate are known. Forexample, the sequences may be formed according to the pioneeringtechniques disclosed in U.S. Pat. No. 5,143,854 (Pirrung et al.), PCT WO92/10092, or U.S. application Ser. No. 08/249,188, now U.S. Pat. No.5,571,639, incorporated herein by reference for all purposes. Theprepared substrates will have a wide range of applications. For example,the substrates may be used for understanding the structure-activityrelationship between different materials or determining the sequence ofan unknown material. The sequence of such unknown material may bedetermined by, for example, a process known as sequencing byhybridization. In one method of sequencing by hybridization, a sequencesof diverse materials are formed at known locations on the surface of asubstrate. A solution containing one or more targets to be sequenced isapplied to the surface of the substrate. The targets will bind orhybridize with only complementary sequences on the substrate.

[0004] The locations at which hybridization occurs can be detected withappropriate detection systems by labeling the targets with a fluorescentdye, radioactive isotope, enzyme, or other marker. Exemplary systems aredescribed in U.S. Pat. No. 5,143,854 (Pirrung et al.) and U.S. patentapplication Ser. No. 08/143,312, also incorporated herein by referencefor all purposes. Information regarding target sequences can beextracted from the data obtained by such detection systems.

[0005] By combining various available technologies, such asphotolithography and fabrication techniques, substantial progress hasbeen made in the fabrication and placement of diverse materials on asubstrate. For example, thousands of different sequences may befabricated on a single substrate of about 1.28 cm² in only a smallfraction of the time required by conventional methods. Such improvementsmake these substrates practical for use in various applications, such asbiomedical research, clinical diagnostics, and other industrial markets,as well as the emerging field of genomics, which focuses on determiningthe relationship between genetic sequences and human physiology.

[0006] As commercialization of such substrates becomes widespread, aneconomically feasible and high-throughput device and method forpackaging the substrates are desired.

SUMMARY OF THE INVENTION

[0007] Methods and devices for packaging a substrate having an array ofprobes fabricated on its surface are disclosed. In some embodiments, abody containing a cavity is provided. A substrate having an array ofprobes is attached to the cavity using, for example, an adhesive. Thebody includes inlets that allow fluids into and through the cavity. Aseal is provided for each inlet to retain the fluid within the cavity.An opening is formed below the cavity to receive a temperaturecontroller for controlling the temperature in the cavity. By forming asealed thermostatically controlled chamber in which fluids can easily beintroduced, a practical medium for sequencing by hybridization isprovided.

[0008] In other embodiments, the body is formed by acoustically weldingtwo pieces together. The concept of assembling the body from two piecesis advantageous. For example, the various features of the package (i.e.,the channels, sealing means, and orientation means) are formed withoutrequiring complex machining or designing. Thus, the packages areproduced at a relatively low cost.

[0009] In connection with one aspect of the invention, a method formaking the chip package is disclosed. In particular, the methodcomprises the steps of first forming a plurality of probe arrays on asubstrate and separating the substrate into a plurality of chips.Typically, each chip contains at least one probe array. A chip is thenmated to a package having a reaction chamber with fluid inlets. Whenmated, the probe array is in fluid communication with the reactionchamber.

[0010] In a specific embodiment, the present invention provides anapparatus for packaging a substrate. The present apparatus includes asubstrate having a first surface and a second surface. The first surfaceincludes a probe array and the second surface is an outer periphery ofthe first surface. The present apparatus also includes a body having amounting surface, an upper surface, and a cavity bounded by the mountingsurface and the upper surface. The second surface is attached to thecavity and the first surface is within the cavity. A cover attached tothe mounting surface for defining an upper boundary to the cavity isalso included. The cavity includes a diffuser and a concentrator. Thediffuser and the concentrator permit laminar fluid flow through thecavity.

[0011] A further understanding of the nature and advantages of theinventions herein may be realized by reference to the remaining portionsof the specification and the attached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012]FIG. 1a illustrates a wafer fabricated with a plurality of probearrays.

[0013]FIG. 1b illustrates a chip.

[0014]FIG. 2a illustrates a scribe and break device.

[0015]FIG. 2b illustrates the wafer mounted on a pick and place frame.

[0016]FIGS. 2c-2 d illustrate the wafer, as displayed by the scribe andbreak device during alignment.

[0017]FIG. 3 illustrates a chip packaging device.

[0018]FIG. 4 illustrates the chip packaging device assembled from twocomponents.

[0019]FIGS. 5a-5 b illustrate the top and bottom view of a top casing ofthe chip packaging device.

[0020]FIG. 5c illustrates a different cavity orientation.

[0021]FIG. 6 illustrates a cross sectional view of the packaging device.

[0022]FIG. 7 illustrates the bottom view of a bottom casing of the chippackaging device.

[0023]FIGS. 8a-8 b illustrate an acoustic welding system.

[0024]FIGS. 9a-9 c illustrate the acoustic welding process used inassembling the chip packaging device.

[0025]FIG. 10 illustrates an adhesive dispensing system used inattaching the chip to the chip packaging device.

[0026] FIGS. 11-13 illustrate in greater detail the adhesive dispensingsystem of FIG. 10.

[0027]FIGS. 14a-14 d illustrate the procedure for aligning the system ofFIG. 10.

[0028]FIGS. 15a-15 e illustrate images obtained during the alignmentprocess of FIGS. 14a-14 d.

[0029]FIGS. 16a-16 b illustrate an alternative embodiment of a packagingdevice.

[0030]FIGS. 17a-17 b illustrate another embodiment of a packagingdevice.

[0031]FIG. 18 illustrates an alternative embodiment for attaching thechip to the packaging device.

[0032]FIG. 19 illustrates another embodiment for attaching the chip tothe packaging device.

[0033]FIGS. 20a-20 b illustrate yet another embodiment for attaching thechip to the packaging device.

[0034]FIG. 21 illustrates an alternative embodiment for attaching thechip to the packaging device.

[0035]FIG. 22 illustrates another embodiment for attaching the chip tothe packaging device.

[0036]FIG. 23 illustrates an alternative embodiment for sealing thecavity on the packaging device.

[0037]FIG. 24 illustrates another alternative embodiment for sealing thecavity on the packaging device.

[0038]FIG. 25 illustrates yet another embodiment for sealing the cavityon the packaging device.

[0039]FIGS. 26a-26 b illustrate an alternative embodiment for sealingthe cavity on the packaging device.

[0040]FIGS. 27a-27 b illustrate an alternative embodiment for mountingthe chip.

[0041]FIG. 28 illustrates an agitation system.

[0042]FIG. 29 illustrates an alternative embodiment of the agitationsystem.

[0043]FIG. 30 illustrates another embodiment of the agitation system.

[0044]FIG. 31 illustrates an alternative embodiment of a chip packagingdevice.

[0045]FIG. 32 illustrates side-views of the chip packaging device ofFIG. 31.

[0046] FIGS. 33-35 illustrate in greater detail the chip packagingdevice of FIG. 31.

[0047]FIG. 36 illustrates a further alternative embodiment of a chippackaging device.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS CONTENTS

[0048] I. Definitions

[0049] II. General

[0050] III. Details of One Embodiment of Invention

[0051] a. Chip Package

[0052] b. Assembly of Chip Package

[0053] c. Chip Attachment

[0054] IV. Details on Alternative Embodiments

[0055] a. Chip Package

[0056] b. Chip Attachment

[0057] c. Fluid Retention

[0058] d. Chip Orientation

[0059] e. Parallel Diagnostics

[0060] V. Details of an Agitation System

[0061] I. Definitions

[0062] The following terms are intended to have the following generalmeanings as they are used herein:

[0063] 1. Probe: A probe is a surface-immobilized molecule that isrecognized by a particular target and is sometimes referred to as aligand. Examples of probes that can be investigated by this inventioninclude, but are not restricted to, agonists and antagonists for cellmembrane receptors, toxins and venoms, viral epitopes, hormones (e.g.,opioid peptides, steroids, etc.), hormone receptors, peptides, enzymes,enzyme substrates, cofactors, drugs, lectins, sugars, oligonucleotidesor nucleic acids, oligosaccharides, proteins, and monoclonal antibodies.

[0064] 2. Target: A target is a molecule that has an affinity for agiven probe and is sometimes referred to as a receptor. Targets may benaturally-occurring or manmade molecules. Also, they can be employed intheir unaltered state or as aggregates with other species. Targets maybe attached, covalently or noncovalently, to a binding member, eitherdirectly or via a specific binding substance. Examples of targets whichcan be employed by this invention include, but are not restricted to,antibodies, cell membrane receptors, monoclonal antibodies and antiserareactive with specific antigenic determinants (such as on viruses, cellsor other materials), drugs, oligonucleotides or nucleic acids, peptides,cofactors, lectins, sugars, polysaccharides, cells, cellular membranes,and organelles. Targets are sometimes referred to in the art asanti-probes or anti-ligands. As the term “targets” is used herein, nodifference in meaning is intended. A “Probe Target Pair” is formed whentwo macromolecules have combined through molecular recognition to form acomplex.

[0065] II. General

[0066] The present invention provides economical and efficient packagingdevices for a substrate having an array of probes fabricated thereon.The probe arrays may be fabricated according to the pioneeringtechniques disclosed in U.S. Pat. No. 5,143,854 (Pirrung et al.), PCT WO92/10092, or U.S. application Ser. No. 08/249,188 filed May 24, 1994,already incorporated herein by reference for all purposes. According toone aspect of the techniques described therein, a plurality of probearrays are immobilized at known locations on a large substrate or wafer.

[0067]FIG. 1a illustrates a wafer 100 on which numerous probe arrays 110are fabricated. The wafer 100 may be composed of a wide range ofmaterial, either biological, nonbiological, organic, inorganic, or acombination of any of these, existing as particles, strands,precipitates, gels, sheets, tubing, spheres, containers, capillaries,pads, slices, films, plates, slides, etc. The wafer may have anyconvenient shape, such as a disc, square, sphere, circle, etc. The waferis preferably flat but may take on a variety of alternative surfaceconfigurations. For example, the wafer may contain raised or depressedregions on which a sample is located. The wafer and its surfacepreferably form a rigid support on which the sample can be formed. Thewafer and its surface are also chosen to provide appropriatelight-absorbing characteristics. For instance, the wafer may be apolymerized Langmuir Blodgett film, functionalized glass, Si, Ge, GaAs,GaP, SiO2, SiN4, modified silicon, or any one of a wide variety of gelsor polymers such as (poly)tetrafluoroethylene,(poly)vinylidenedifluoride, polystyrene, polycarbonate, or combinationsthereof. Other materials with which the wafer can be composed of will bereadily apparent to those skilled in the art upon review of thisdisclosure. In a preferred embodiment, the wafer is flat glass orsingle-crystal silicon.

[0068] Surfaces on the solid wafer will usually, though not always, becomposed of the same material as the wafer. Thus, the surface may becomposed of any of a wide variety of materials, for example, polymers,plastics, resins, polysaccharides, silica or silica-based materials,carbon, metals, inorganic glasses, membranes, or any of the above-listedwafer materials.

[0069] Wafer 100 includes a plurality of marks 145 that are located instreets 150 (area adjacent to the probe arrays). Such marks may be usedfor aligning the masks during the probe fabrication process. In effect,the marks identify the location at which each array 110 is to befabricated. The probe arrays may be formed in any geometric shape. Insome embodiments, the shape of the array may be squared to minimizewasted wafer area. After the probe arrays have been fabricated, thewafer is separated into smaller units known as chips. The wafer, forexample, may be about 5×5 inches on which 16 probe arrays, eachoccupying an area of about 12.8 cm², are fabricated.

[0070]FIG. 1b illustrates a chip that has been separated from the wafer.As illustrated, chip 120 contains a probe array 110 and a plurality ofalignment marks 145. The marks serve multiple functions, such as: 1)aligning the masks for fabricating the probe arrays, 2) aligning thescriber for separating the wafer into chips, and 3) aligning the chip tothe package during the attachment process. In some embodiments, suchchips may be of the type known as Very Large Scale Immobilized PolymerSynthesis (VLSIPS™) chips.

[0071] According to a specific embodiment, the chip contains an array ofgenetic probes, such as an array of diverse RNA or DNA probes. In someembodiments, the probe array will be designed to detect or study agenetic tendency, characteristic, or disease. For example, the probearray may be designed to detect or identify genetic diseases such ascystic fibrosis or certain cancers (such as P53 gene relevant to somecancers), as disclosed in U.S. patent application Ser. No. 08/143,312,already incorporated by reference.

[0072] According to one embodiment, the wafer is separated into aplurality of chips using a technique known as scribe and break. FIG. 2aillustrates a fully programmable computer controlled scribe and breakdevice, which in some embodiments is a DX-III Scriber breakermanufactured by Dynatex International™. As shown, the device 200includes a base 205 with a rotation stage 220 on which a wafer ismounted. The rotation stage includes a vacuum chuck for fixing the waferthereon. A stepper motor, which is controlled by the system, rotatesstage 220. Located above the stage is a head unit 230 that includes acamera 232 and cutter 231. Head unit 230 is mounted on a dual-axisframe. The camera generates an image of the wafer on video display 210.The video display 210 includes a cross hair alignment mark 215. Thecamera, which includes a zoom lens and a fiber optic light, allows auser to inspect the wafer on the video display 210. A control panel 240is located on the base for operating device 200.

[0073] In operation, a user places a wafer 100 on a frame 210 asillustrated in FIG. 2b. The surface of frame 210 is composed of aflexible and sticky material. The tackiness of the frame prevents thechips from being dispersed and damaged during the breaking process.Frame 210 may be a pick and place frame or a hoop that is commonlyassociated with fabrication of semiconductors. Referring back to FIG.2a, a user places the frame with the wafer on the rotation stage 220. Insome embodiments, the frame is held on the rotation stage by vacuumpressure. The user then aligns the wafer by examining the imagedisplayed on the video display 210.

[0074] According to one embodiment, wafer alignment is achieved in twosteps. First, using the control panel 240, the user rotates stage 220.The stage is rotated until streets 150 are aligned with the cross hair215 on the display, as illustrated in FIG. 2c. Next, the user moves thecutter until it is aligned at the center of one of the streets. Thisstep is performed by aligning horizontal line 216 of the cross hairbetween alignment marks 145, as shown in FIG. 2d.

[0075] Once the cutter is aligned, the user instructs the device toscribe the wafer. In some embodiments, various options are available tothe user, such as scribe angle, scribe pressure, and scribe depth. Theseparameters will vary depending on the composition and/or thickness ofthe wafer. Preferably, the parameters are set to scribe and break thewafer without causing any damage thereto or penetrating through theframe. The device repeatedly scribes the wafer until all the streets inone axis have been scribed, which in one embodiment is repeated 5 times(a 4×4 matrix of probe arrays). The user then rotates the stage 90° toscribe the perpendicular streets.

[0076] Once the wafer has been scribed, the user instructs the device tobreak or separate the wafer into chips. Referring back to FIG. 2a, thedevice 200 breaks the wafer by striking it beneath the scribe with animpulse bar located under the rotation table 220. The shock from theimpulse bar fractures the wafer along the scribe. Since most of theforce is dissipated along the scribe, device 200 is able to produce highbreaking forces without exerting significant forces on the wafer. Thus,the chips are separated without causing any damage to the wafer. Onceseparated, the chips are then packaged. Of course, other moreconventional techniques, such as the sawing technique disclosed in U.SPat. No. 4,016,855, incorporated herein by reference for all purposes,may be employed.

[0077] III. Details of One Embodiment of the Invention

[0078] a. Chip Package

[0079]FIG. 3 illustrates a device for packaging the chips. Package 300contains a cavity 310 on which a chip is mounted. The package includesinlets 350 and 360 which communicate with cavity 310. Fluids arecirculated through the cavity via inlets 350 and 360. A septum, plug, orother seal may be employed to seal the fluids in the cavity. Alignmentholes 330 and 335 may be provided for alignment purposes. In someembodiments, the package may include a non-flush edge 320. In somedetection systems, the packages may be inserted into a holder similar toan audio cassette tape. The asymmetrical design of the package willassure correct package orientation when inserted into the holder.

[0080]FIG. 4 illustrates one embodiment of the package. As shown in FIG.4, the chip package is manufactured by mating two substantiallycomplementary casings 410 and 420 to form finished assembly 300.Preferably, casings 410 and 420 are made from injection molded plastic.Injection molding enables the casings to be formed inexpensively. Also,assembling the package from two parts simplifies the construction ofvarious features, such as the internal channels for introducing fluidsinto the cavity. As a result, the packages may be manufactured at arelatively low cost.

[0081]FIGS. 5a-5 b show the top casing 410 in greater detail. FIG. 5ashows a top view and FIG. 5b shows a bottom view. Referring to FIG. 5a,top casing 410 includes an external planar surface 501 having a cavity310 therein. In some embodiments, the surface area of casing 410sufficiently accommodates the cavity. Preferably, the top casing is ofsufficient size to accommodate identification labels or bar codes inaddition to the cavity. In a specific embodiment, the top casing isabout 1.5″ wide, 2″ long, and 0.2″ high.

[0082] Cavity 310 is usually, though not always, located substantiallyat the center of surface 501. The cavity may have any conceivable size,shape, or orientation. Preferably, the cavity is slightly smaller thanthe surface area of the chip to be placed thereon and has a volumesufficient to perform hybridization. In one embodiment, the cavity maybe about 0.58″ wide, 0.58″ long, and 0.2″ deep.

[0083] Cavity 310 may include inlets 350 and 360. Selected fluids areintroduced into and out of the cavity via the inlets. In someembodiments, the inlets are located at opposite ends of the cavity. Thisconfiguration improves fluid circulation and regulation of bubbleformation in the cavity. The bubbles agitate the fluid, increasing thehybridization rate between the targets and complementary probesequences. In one embodiment, the inlets are located at the top andbottom end of the cavity when the package is oriented vertically such asat the opposite comers of the cavity. Locating the inlet at the highestand lowest positions in the cavity facilitates the removal of bubblesfrom the cavity.

[0084]FIG. 5c illustrates an alternative embodiment in which cavity 310is oriented such that the edges of the cavity 310 and the casing 410 arenon-parallel. This configuration allows inlets 350 and 360 to besituated at the absolute highest and lowest locations in the cavity whenthe package is vertically oriented. As a result, bubbles or fluiddroplets are prevented from being potentially trapped in the cavity.

[0085] Referring back to FIG. 5a, a depression 550 surrounds the cavity.In some embodiments, a ridge 560 may be provided at the edge of thedepression so as to form a trough. The ridge serves to support the chipabove the cavity. To attach the chip to the package, an adhesive may bedeposited in the trough. This configuration promotes efficient use ofchip surface area, thus increasing the number of chips yielded from awafer.

[0086] Top casing 410 includes alignment holes 330 and 335. In someembodiments, holes 330 and 335 are different in size to ensure correctorientation of the package when mounted on an alignment table.Alternatively, the holes may have different shapes to achieve thisobjective. Optionally, the holes taper radially inward from surface 501toward 502 to reduce the friction against alignment pins while stillmaintaining adequate contact to prevent slippage.

[0087] Referring to FIG. 5b, channels 551 and 561 are optionally formedon internal surface 502. Channels 551 and 561 communicate with inlets350 and 360 respectively. A depression 590 is formed below cavity.According to some embodiments, the shape of depression 590 issymmetrical to the cavity with exception to corners 595 and 596, whichaccommodate the inlets. The depth of depression 590 may be, for example,about 0.7″. As a result, the bottom wall of the cavity is about 0.05″thick. Depression 590 may receive a temperature controller to monitorand maintain the cavity at the desired temperature. By separating thetemperature controller and cavity with a minimum amount of material, thetemperature within the cavity may be controlled more efficiently andaccurately. Alternatively, channels may be formed on surface 502 forcirculating air or water to control the temperature within the cavity.

[0088] In some embodiments, certain portions 595 of internal surface 502may be eliminated or cored without interfering with the structuralintegrity of the package when assembled. Coring the casing reduces thewall thickness, causing less heat to be retained during the injectionmolding process; potential shrinkage or warpage of the casing issignificantly reduced. Also, coring decreases the time required to coolthe casing during the manufacturing process. Thus, manufacturingefficiency is improved.

[0089] In one embodiment, the top casing and bottom casing are matedtogether using a technique known as acoustic or ultrasonic welding.Accordingly, “energy directors” 510 are provided. Energy directors areraised ridges or points, preferably v-shaped, that are used in anacoustic welding process. The energy directors are strategicallylocated, for example, to seal the channels without interfering withother features of the package and to provide an adequate bond betweenthe two casings. Alternatively, the casings may be mated together byscrews, glue, clips, or other mating techniques.

[0090] FIGS. 6 shows a cross sectional view of the cavity 310 with chip120 mounted thereon in detail. As shown, a depression 550 is formedaround cavity 310. The depression includes a ridge 560 which supportschip 120. The ridge and the depression create a trough around cavity310. In some embodiments, the trough is sufficiently large to receive anadhesive 630 for attaching the chip to the package. In one embodiment,the trough is about 0.08″ wide and 0.06″ deep. When mounted, the edge ofthe chip protrudes slightly beyond ridge 550, but without contactingside 625 of the depression. This configuration permits the adhesive tobe dispensed onto the trough and provides adequate surface area for theadhesive to attach chip 120 to the package.

[0091] According to some embodiments, the back surface 130 of chip 120is at least flush or below the plane formed by surface 501 of casing410. As a result, chip 120 is shielded by surface 501 from potentialdamage. This configuration also allows the packages to be easily storedwith minimal storage area since the surfaces are substantially flat.

[0092] Optionally, the bottom of the cavity includes a light absorptivematerial, such as a glass filter or carbon dye, to prevent impinginglight from being scattered or reflected during imaging by detectionsystems. This feature improves the signal-to-noise ratio of such systemsby significantly reducing the potential imaging of undesired reflectedlight.

[0093]FIG. 7 shows the internal surface of bottom casing 420 in greaterdetail. As shown, the bottom casing 420 is substantially planar andcontains an opening 760 therein. Preferably, the casing 420 is slightlywider or slightly longer than the top casing. In one embodiment, casing420 is about 1.6″ wide, 2.0″ long, and 0.1″ deep, which creates anon-flush edge on the finish assembly. As previously mentioned, thisdesign ensures that the package is correctly oriented when mounted ontothe detection systems.

[0094] In some embodiments, opening 760 is spatially located at aboutthe depression below the cavity. The opening also has substantially thesame geometric configuration as the depression to allow the temperaturecontroller to contact as much of the bottom of the cavity as possible.

[0095] Internal surface 701 of casing 420 includes depressions 730 and740. A port 731 is located in depression 730 and a port 741 is locatedin depression 740. Ports 731 and 741 communicate with channels on thetop casing (350 and 360 in FIG. 5b) when the package is assembled. Aseal 790, which may be a septum composed of rubber, teflon/rubberlaminate, or other sealing material is provided for each depression. Theseptum may be of the type commonly used to seal and reseal vessels whena needle is inserted into the septum for addition/removal of fluids. Theseptums, when seated in the depressions, extend slightly above surface,which in some embodiments is about 0.01.

[0096] This design causes casings 410 and 420 to exert pressure on theseptum, forming a seal between the ports and the channels. The seal ismaintained even after fluid is injected into the cavity since thepressure immediately forces the septum to reseal itself after the needleor other fluid injecting means is removed from the port. Thus, anefficient and economical seal for retaining fluid in the cavity isprovided.

[0097] Also, casing 420 includes the complementary half alignment holes330 and 335, each tapering radially inward from the external surface.Further, certain areas 765 on internal surface 701 may be cored, assimilar to the internal surface of the top casing.

[0098]FIG. 31 is a simplified illustration of an alternative embodimentof a chip packaging device 3100 according to the present invention. Thechip packaging device includes a plurality of casings 3200, 3300, and3400. The casings may be defined as a top casing 3200, a middle casing3300, and a bottom casing 3400. The casings are made of known plasticmaterials such as ABS plastic, polyvinylchloride, polyethylene, productssold under the trademarks TEFLON™ and KALREZ™ and the like, amongothers. Preferably, the casings can be made by way of injection moldingand the like. Assembling the chip packaging device from three casingssimplifies construction for the fabrication of internal channels and thelike, and can also be made at a relatively low cost.

[0099] Support structures (or alignment holes) exist at selectedlocations of the chip packing device. The support structures can be usedto mount or position the chip packaging device to an apparatus, e.g.,scanner or the like. In an embodiment, the top casing 3200 includessupport structures 3201 and 3203 on each side of a center opening 3209.The middle casing 3300 includes similar support structures 3313 and 3315which are complementary to the support structures 3201 and 3203,respectively, in the top casing. The bottom casing also includes similarsupport structures 3403 and 3401, respectively, which are complementaryto the support structures in the top casing and the middle casing. Asshown, each of the support structures on each side of the center openingalign with each other. Each support structure is, for example, anaperture through the casing. The aperture includes an outer peripherydefined by a geometrical shape which may be round, rectangular,trapezoidal, hexagonal, or the like.

[0100] The present chip packaging device assembles with use ofcomplementary alignment pins and bores on the casings. By way ofalignment pins (not shown), the top casing aligns with and inserts intoalignment bores 3301, 3303 in the middle casing 3300. Alternatively, themiddle casing can have alignment pins or the like and the top casing hasthe alignment bores or the like. The bottom casing includes alignmentpins 3407 and 3409 which align to and insert into alignment bores (notshown) in bottom portions of the middle casing. The use of alignmentbores and pins provide for ease in assembly of the chip carrier. Uponassembly, the alignment bores and pins on the casings prevent thecasings from moving laterally relative to each other.

[0101] A center opening 3209 in the top casing overlies a center portion3317 of the middle casing 3300. The center portion 3317 of the middlecasing includes an inner annular region (or cavity edges) with a bottomportion which is preferably a flat bottom portion. The flat bottomportion of the middle casing and portions of the bottom casing includingedges define a cavity 3405. A chip is placed overlying an underlyingportion of the cavity 3407.

[0102] Optionally, a temperature control mechanism such as a heater, acooler, or a combination thereof is disposed into the center openingagainst the bottom portion of the middle casing. The temperature controlmechanism can be any suitable thermally controlled element such as aresistive element, a temperature controlled block or mass,thermoelectric modules, or the like. The temperature control mechanismtransfers heat via conduction to the bottom center portion, whichtransfers heat to, for example, fluid in the cavity or the chip.Alternatively, the temperature control mechanism sinks heat away from,for example, fluid in the cavity or the chip through the bottom centerportion. The temperature control mechanism maintains a selectedtemperature in the cavity. The temperature control mechanism alsoincludes a temperature detection device such as a thermocouple whichprovides signals corresponding to temperature readings. A controllerreceives the signals corresponding to the temperature readings, andadjusts power output to the temperature control mechanism to maintainthe selected temperature.

[0103] The top casing 3200 also includes channels 3205 and 3207 forfluid transfer. The channels 3205 and 3207 communicate with annularregions 3309 and 3311, respectively, on the middle casing 3300 for fluidtransfer. A septum, a plug, an o-ring, a gasket, or the like via annularregions 3309 and 3311 seals fluids within the top casing channels 3205and 3207 and the middle casing. The bottom casing includes channels 3411and 3413 in communication with channels 3307 and 3305, respectively. Aseptum, a plug, an o-ring, a gasket, or the like seals the fluids withinthe bottom casing channels 3411 and 3413 and the middle casing channels3305 and 3307.

[0104] The chip packaging device provides an even distribution of fluid(or fluid flow) through the cavity over a top surface (or inner oractive surface) of the chip. For example, a selected fluid enterschannel 3207, flows through channel 3307, changes direction and flowsthrough channel 3411, and evenly distributes into the cavity 3405 overthe top surface of the chip. As previously noted, the cavity is definedby flat bottom portion and cavity edges. A selected fluid exits thecavity by way of channel 3413, channel 3305, and channel 3205. The fluidflow over the top surface of the chip is preferably laminar, but mayalso be turbulent, a combination thereof or the like. By way of thepresent chip packaging device, a substantial portion of turbulent flowremains at an upper portion of the channel 3411, and does not enter thecavity.

[0105] Preferably, a selected fluid enters the cavity by way of channel3205, channel 3305, and channel 3413. The selected fluid exits thecavity through channel 3411, channel 3307, and channel 3207. In apreferred embodiment, the fluid flows against the direction of gravitythrough the cavity. Of course, other fluid flow routes may also beemployed depending upon the particular application.

[0106]FIG. 32 illustrates an assembled chip packaging device 3100according to the present invention. As shown are a top-view 3200, aside-view 3500, a bottom-view 3400, and a front-view 3600 of theassembled chip packaging device 3100. The assembled chip packagingdevice 3100 includes the bottom casing 3400, the middle casing 3300, andthe top casing 3200.

[0107] The top-view 3200 of the top casing includes alignment structures3205, 3215 surrounding opening 3209. The opening 3209 includes abevelled annular region 3211 surrounding the periphery of the channel3209. The alignment bores 3203 and 3201 also include bevelled annularregions 3213 and 3215, respectively. A bevelled annular region 3217,3221 also surrounds each fluid channel 3205, 3207 to assist with fluidflow therethrough.

[0108] The bottom-view 3400 of the bottom casing includes alignmentstructures 3401, 3403 surrounding the cavity 3405. The cavity includes aflat bottom peripheral portion 3415, a bevelled portion 3417 extendingfrom the flat bottom peripheral portion, and a flat upper portion 3419surrounding the bevelled portion. The chip includes an outer peripherywhich rests against the flat bottom peripheral portion 3415. Thebevelled portion aligns the chip onto the flat bottom peripheral portion3415. Similar to the previous embodiments, the top casing extendsoutside 3421 the middle and bottom casings.

[0109] The cavity 3405 is preferably located at a center of the bottomcasing, but may also be at other locations. The cavity may be round,square, rectangular, or any other shape, and orientation. The cavity ispreferably smaller than the surface area of the chip to be placedthereon, and has a volume sufficient to perform hybridization and thelike. In one embodiment, the cavity includes dimensions such as a lengthof about 0.6 inch, a width of about 0.6 inch and a depth of about 0.07inch.

[0110] In a preferred embodiment, the bottom casing with selected cavitydimensions may be removed from the middle and top casings, and replacedwith another bottom casing with different cavity dimensions. This allowsa user to attach a chip having a different size or shape by changing thebottom casing, thereby providing ease in using different chip sizes,shapes, and the like. Of course, the size, shape, and orientation of thecavity will depend upon the particular application.

[0111] FIGS. 33-35 illustrate in greater detail the chip packagingdevice of FIG. 31. FIG. 33 illustrates simplified top-view 3260 andbottom-view 3250 diagrams of the top casing 3200. As shown, thereference numerals refer to the same elements as the top casing of FIG.31. FIG. 34 illustrates a simplified top-view 3350 and bottom-view 3360diagrams of the middle casing 3300. As shown, the reference numeralsrefer to the same elements as the middle casing of FIG. 31. In addition,the bottom-view of the casing includes a substantially smooth and planarbottom surface 3361. A portion of the bottom surface defines an upperportion of the cavity. But the bottom surface can also be textured,ridged, or the like to create turbulence or a selected fluid flowthrough the cavity. The bottom surface is preferably a hydrophobicsurface which enhances laminar flow through the cavity. Of course, thetype of bottom surface depends upon the particular application.

[0112]FIG. 35 illustrates simplified top-view 3460 and bottom-view 3450diagrams of the bottom casing 3400. As shown, the reference numeralsrefer to the same elements as the bottom casing of FIG. 31. In anembodiment, fluid from channel 3305 changes direction at an upperportion 3431 of the channel and flows to a lower portion 3433 of thechannel. Fluid evenly distributes from the lower portion 3433 via afluid distribution point 3435. The distributed fluid evenly passes overa slanted edge (or bevelled edge) 3437 which drops fluid evenly to a topsurface of the chip in the cavity. By way of slanted edge 3427 whichslopes up to a fluid concentration point 3425, fluid leaves the cavityand enters the channel 3411. In particular, fluid leaves the cavity andenters a lower portion 3423 of the channel, flows through the channel,and changes directions at an upper portion 3421 of the channel. Eachchannel includes a length L and a width W. The distribution point andthe concentration point are positioned at a distance away from thecavity to substantially prevent turbulence from forming in the cavity,and in particular over the top surface of the chip. The channels areeach angled at an angle Θ ranging from about 2 degrees to about 90degrees, but is preferably about 5 degrees to about 45 degrees. Theangle enhances an even distribution of laminar flow into the cavity. Ofcourse, the exact angle, channel shape, and dimensions depend upon theparticular application.

[0113]FIG. 36 illustrates a simplified cross-sectional view of analternative embodiment 3600 of the chip packaging device. The chippackaging device includes the three casings 3200, 3300, and 3400 of theprevious embodiment, and also includes hollow pins, needles, or the like3601 and 3603. Each of the pins transfers a selected fluid to and fromthe cavity 3405. Preferably, each pin 3601 includes an external opening3609, a tubular region 3611, an inner opening 3607, a pointed tip 3605,and other elements. The pin is made from a suitable material such as aglass, a stainless steel or any other high quality material to transferfluids to and from the cavity 3405.

[0114] In a preferred embodiment, each pin is inserted into its channelregion 3205 or 3207. A point on the pin tip pierces through, forexample, a septum at an annular region 3309 or 3311. A selected fluidtravels through pin 3603 (through channel 3205 and at least a portion of3305), enters the upper region of channel 3413, and into the cavity3405. The selected fluid travels from the cavity, through pin 3601, andto the external apparatus. Alternatively, the selected fluid enters thecavity via pin 3601 and exits the cavity via pin 3603. The selectedfluid may also enter the cavity via pin and exit the cavity through thechannels without use of a pin. The selected fluid may further enter thecavity through the channels without use of a pin and exit through a pin.Of course, the particular pin used and fluid flow will depend upon theapplication.

[0115] It should be noted that the even distribution of fluid flowthrough the cavity prevents “hot spots” from occurring in the cavity.For example, the even distribution of fluid through the cavity by way ofthe previous embodiment substantially prevents fluid from becomingsubstantially turbulent at certain locations. This prevents “hot spots”caused by such turbulent fluid. The hot spots are often caused by higherchemical activity or exothermic reactions and the like by way ofturbulence in such certain locations.

[0116] b. Assembly of Chip Package

[0117] According to one embodiment, the top and bottom casing areattached by a technique known as ultrasonic or acoustic welding. FIG. 8ais a schematic diagram of acoustic welding system used for assemblingthe package. In some embodiments, the welding system 800 is a HS Dialogultrasonic welder manufactured by Herrmann Ultrasonics Inc. System 800includes a platform 850 mounted on base 810. Platform 850 accommodatesthe top and bottom casings during the assembling process.

[0118] An acoustic horn 860 is mounted on a frame above platform 850.The horn translates vertically (toward and away from platform 850) onthe frame by air pressure. The horn is connected to a frequencygenerator 870, which in some embodiments is a 20 KHz generatormanufactured by Herrmann Ultrasonics Inc. System 800 is controlled by acontroller 880, which, for example, may be a Dialog 2012 manufactured byHerrmann Ultrasonics Inc. Controller 880 may be configured to acceptcommands from a digital computer system 890. Computer 890 may be anyappropriately programmed digital computer of the type that is well knownto those skilled in the art such as a Gateway 486DX operating at 33 MHz.

[0119]FIG. 8b illustrates platform 850 in greater detail. The platform850 is substantially planar and includes alignment pins 851 and 852.Alignment pins 851 and 852 are used to align both the top and bottomcasings during the welding process. In some embodiments, a pad 890,which may be composed of silicone rubber or other energy absorbingmaterial, is located on platform 850 to prevent damage to the packageduring assembly.

[0120]FIG. 9a illustrates the acoustic welding system in operation. Asshown, bottom casing 420, having a septum 790 seated in each depression,is mounted onto platform table 850 and held in place by alignment pins.Top casing 410 is then aligned above the bottom casing with alignmentpins. The system then commences the welding process by lowering horn 860until it contacts the top surface of casing 410.

[0121]FIG. 9b illustrates the casing and horn in detail. As shown, thehorn 860 presses against top casing 410, thereby forcing energydirectors 510 to interface with bottom casing 420. The system thenactivates the frequency generator, causing the welding horn to vibrate.

[0122]FIG. 9c illustrates in detail the energy directors during thewelding process. As shown in step 9001, welding horn 860 forces energydirectors 510 against bottom casing 420. At step 9002, the systemvibrates the welding horn, which in some embodiments is at 20 KHz. Theenergy generated by the horn melts the energy directors. Simultaneously,the horn translates downward against the package. At step 9003, thepressure exerted by the horn causes the energy directors to fuse withthe bottom casing. At step 9004, the welding process is completed whenthe horn reaches its weld depth, for example, of about 0.01″. Of course,the various welding parameters may be varied, according to thecomposition of the materials used, to achieve optimum results.

[0123] c. Chip Attachment

[0124] According to some embodiments, an ultraviolet cured adhesiveattaches the chip to the package. FIG. 10 schematically illustrates anadhesive dispensing system used in attaching the chip. The dispensingsystem 1000 includes an attachment table 1040 to accommodate the packageduring the attachment process. A chip alignment table 1050 for aligningthe chip is located adjacent to attachment table 1040. A head unit 1030for dispensing the adhesive is located above tables 1040 and 1050. Thehead unit 1030 also includes a camera that generates an output to videodisplay 1070. Video display 1070, in some embodiments, includes a crosshair alignment mark 1071. The head unit is mounted on a dual-axis (x-y)frame for positioning during alignment and attachment of the chip. Theoperation of the dispensing system is controlled by a computer 1060,which in some embodiments may be Gateway 486DX operating at 33 MHz.

[0125]FIG. 11 illustrates the attachment table in greater detail. Theattachment table 1040 has a substantially flat platform 1110 supportedby a plurality of legs 1105. Alignment pins 1115 and 1116, which securethe package during the attachment process, are located on the surface ofplatform 1110.

[0126] Optionally, a needle 1120 is provided. Needle 1120 includes achannel 1121 and is connected to a vacuum pump. In operation, the needleis inserted into one of the ports of the package in order to generate avacuum in the cavity. The vacuum pressure secures the chip to thepackage during the attachment process.

[0127]FIG. 12a shows table 1050 in greater detail. Table 1050 includes asubstantially flat platform 1210 having a depression 1240 for holding achip. In some embodiments, a port 1241 is provided in depression 1240.Port 1241 is connected to a vacuum pump which creates a vacuum in thedepression for immobilizing the chip therein. Platform 1210 is mountedon a combination linear rotary stage 1246, which in some embodiments maybe a model 26LR manufactured by DARDAL, and a single axis translationstage 1245, which may be a model CR2226HSE2 manufactured by DARDAL.

[0128]FIG. 12b illustrates depression 1240 in greater detail. As shown,a ledge 1241 surrounds the depression 1240. Ledge 1241 supports the chipwhen it is placed above depression 1240. Since the chips are placed overthe depression with the probes facing the table, this design protectsthe probes from being potentially damaged during alignment.

[0129]FIG. 13 illustrates the head unit 1030 in greater detail. Asshown, the head unit 1030 includes a camera assembly 1320 that generatesan output to a video display. A light 1360 is provided to enable thecamera to focus and image an object of interest. The head unit alsoincludes an ultraviolet light 1350 for curing the adhesive, a vacuumpickup 1330 for moving chip during the attachment process, and anadhesive dispenser 1340.

[0130] In operation, a chip package is placed onto table 1040. Aspreviously described, the alignment pins on the table immobilize thepackage. The user begins the chip attachment process by calibrating thehead unit. This may be done by moving the camera above the package andaligning it with a mark on the package, as shown in FIG. 14a. Forconvenience, one of the alignment pins may be used as an alignment mark.FIG. 14b illustrates a typical image 1440 generated by the camera duringthis step. As shown, the head unit is not aligned with pin 1480. Toalign the head unit, the user translates it in both the x and ydirection until pin 1480 is located at the intersection 1477 of thecross hair on the video display, as illustrated in FIG. 14c.

[0131] Next, the chip is inserted into the depression on the chipalignment table. FIG. 14c is a flow chart indicating the steps foraligning the chip. At step 1410, the system positions the camera (headunit) above one of the chip's alignment marks. The camera images thealignment mark on the video display. At this point, the mark is normallymisaligned (i.e., the mark is not located at the intersection of thecross hair alignment mark). At step 1420, the user adjusts the chipalignment table in both the x and y direction until the mark issubstantially located at the intersection of the cross hair. Since norotational adjustments were made, the mark may be misaligned angularly.

[0132] At step 1430, the user instructs the system to move the cameraabove a second alignment mark, which usually is at an opposite corner ofthe chip. Again, an image of the alignment mark is displayed. At thisstage, the alignment mark is probably misaligned in the x, y, andangular directions. At step 1440, the user adjusts the rotational stage,x-stage, and y-stage, if necessary, to align the mark with the crosshair on the video display. In instances where the rotational stage hasbeen rotated, the first alignment mark will become slightly misaligned.To compensate for this shift, the user repeats the alignment processbeginning at step 1450 until both marks are aligned. Of course, imageprocessing techniques may be applied for automated head unit and chipalignment.

[0133]FIG. 15a is an example of an image displayed by the video screenduring step 1410. As shown, the first alignment mark (lower left cornerof the chip) is not aligned with the cross hair marking. FIG. 15bexemplifies an image of the first alignment mark after adjustments weremade by the user. FIG. 15c illustrates a typical image displayed byvideo screen during step 1430. As illustrated, the second alignment mark(upper right corner of the chip) is misaligned in the x, y, and angulardirections. FIG. 15d illustrates an image of the second mark followinginitial adjustments by the user at step 1440. FIG. 15e illustrates theorientation of the second alignment mark after the chip has beenaligned.

[0134] Once the chip is aligned, the vacuum holding the chip on theattachment table is released. Thereafter, the pickup on the head unitremoves the chip from the table and aligns it on the cavity of thepackage. In some embodiments, the chip is mated to the pickup by avacuum.

[0135] Optionally, the user may check to ensure that the chip iscorrectly aligned on the cavity by examining the chip's alignment markswith the camera. If the chip is out of position, the chip is removed andrealigned on the alignment table. If the chip is correctly positioned,the system deposits an adhesive by moving the dispenser along the troughsurrounding the cavity. In some embodiments, the vacuum is releasedbefore depositing the adhesive in the trough. This step is merelyprecautionary and implemented to ensure that the vacuum does not causeany adhesive to seep into the cavity. Once the adhesive is deposited,the system reexamines the chip to determine if the adhesive had movedthe chip out of position. If the chip is still aligned, the head unitlocates the ultraviolet light above the adhesive and cures it for a timesufficient to harden the adhesive, which in one embodiment is about 10seconds. Otherwise, the chip is realigned.

[0136] Upon completion, the chip package will have a variety of uses.For example, the chip package will be useful in sequencing geneticmaterial by hybridization. In sequencing by hybridization, the chippackage is mounted on a hybridization station where it is connected to afluid delivery system. Such system is connected to the package byinserting needles into the ports and puncturing the septums therein. Inthis manner, various fluids are introduced into the cavity forcontacting the probes during the hybridization process.

[0137] Usually, hybridization is performed by first exposing the samplewith a prehybridization solution. Next, the sample is incubated underbinding conditions with a solution containing targets for a suitablebinding period. Binding conditions will vary depending on theapplication and are selected in accordance with the general bindingmethods known including those referred to in: Maniatis et al., MolecularCloning: A Laboratory Manual (1989), 2nd Ed., Cold Spring Harbor, N.Y.and Berger and Kimmel, Methods in Enzymology, Volume 152, Guide toMolecular Cloning Techniques (1987), Academic Press, Inc., San Diego,Calif.; Young and Davis (1983) Proc. Natl. Acad. Sci. U.S.A.) 80: 1194,which are incorporated herein by reference. In some embodiments, hesolution may contain about 1 molar of salt and about 1 to 50 nanomolarof targets. Optionally, the fluid delivery system includes an agitatorto improve mixing in the cavity, which shortens the incubation period.Finally, the sample is washed with a buffer, which may be 6× SSPEbuffer, to remove the unbound targets. In some embodiments, the cavityis filled with the buffer after washing the sample.

[0138] Thereafter, the package may be aligned on a detection or imagingsystem, such as those disclosed in U.S. Pat. No. 5,143,854 (Pirrung etal.) or U.S. patent application Ser. No. 08/495,889 (Attorney DocketNumber 11509-117), already incorporated herein by reference for allpurposes. Such detection systems may take advantage of the package'sasymmetry (i.e., non-flush edge) by employing a holder to match theshape of the package specifically. Thus, the package is assured of beingproperly oriented and aligned for scanning. The imaging systems arecapable of qualitatively analyzing the reaction between the probes andtargets. Based on this analysis, sequence information of the targets isextracted.

[0139] IV. Details on Alternative Embodiments

[0140] a. Chip Package Orientation

[0141]FIGS. 16a-16 b illustrate an alternative embodiment of thepackage. FIG. 16a shows a top view and FIG. 16b shows a bottom view. Asshown in FIG. 16a, a cavity 1620 is located on a top surface 1610 of thepackage body 1600. The body includes alignment holes 1621 and 1622 thatare used, for example, in mating the chip to the package. Optionally, aplurality of ridges 1690 is located at end 1660 of the body. Thefriction created by ridges 1690 allows the package to be handled easilywithout slippage.

[0142] The body also includes two substantially parallel edges 1630 and1640. As shown, edge 1640 is narrowed at end 1665 to create an unevenedge 1645. The asymmetrical design of the body facilitates correctorientation when mounted onto detection systems. For example, detectionsystems may contain a holder, similar to that of an audio cassette tape,in which end 1665 is inserted.

[0143] Referring to FIG. 16b, ports 1670 and 1671 communicate withcavity 1620. A seal is provided for each port to retain fluids in thecavity. Similar to the op surface, the bottom surface may optionallyinclude a plurality of ridges 1690 at end 1660.

[0144]FIGS. 17a-17 b illustrate an alternative embodiment of thepackage. FIG. 17a shows a top view and FIG. 17b shows a bottom view.Referring to FIG. 17a, a cavity 1720 is located on a top surface 1710 ofthe package body 1700. The body may be formed in the shape of a diskwith two substantially parallel edges 1730 and 1740. Alignment holes1721 and 1722, which may be different in size or shape, are located onthe body. In some embodiments, the package is inserted like an audiocassette tape into detection systems in a direction parallel to edges1730 and 1740. Edges 1730 and 1740 and alignment holes prevent thepackage from being inserted incorrectly into the detection systems.

[0145] As shown in FIG. 17b, ports 1730 and 1740 are located on thebottom surface 1715 of the package. Ports 1730 and 1740 communicate withcavity 1720 and each include a seal 1780 for sealing fluids in thecavity.

[0146] b. Chip Attachment

[0147]FIG. 18 illustrates an alternative embodiment for attaching thechip to the package. As shown, two concentric ledges 1810 and 1820surround the perimeter of cavity 310. Ledge 1820 supports the chip 120when mounted above cavity 310. Ledge 1810, which extends beyond chip120, receives an adhesive 1860 such as ultraviolet cured silicone,cement, or other adhesive for attaching the chip thereto.

[0148]FIG. 19 illustrates another embodiment for attaching the chip tothe package. According to this embodiment, a ledge 1910 is formed aroundcavity 310. Preferably, the ledge is sufficiently large to accommodatean adhesive 1920 such as an adhesive film, adhesive layer, tape, or anyother adhesive layer. Chip 120 attaches to the package when it contactsthe adhesive film.

[0149]FIG. 20a illustrates yet another embodiment for attaching a chipto the package. As shown, a clamp 2010, such as a frame having aplurality of fingers 2015, attaches the chip to the package. FIG. 20billustrates a cross sectional view. A ridge 2020 on surface 501surrounds cavity 310. The ridge includes a ledge 2025 upon which chip120 rests. Optionally, a gasket or a seal 2070 is located between theledge and chip to ensure a tight seal around cavity 310. Clamp 2010 isattached to side 2040 of ridge 2020 and surface 501. In someembodiments, clamp 2010 is acoustically welded to the body. Accordingly,clamp 2010 includes energy directors 2050 located at its bottom.Alternatively, screws, clips, adhesives, or other attachment techniquesmay be used to mate clamp 2010 to the package. When mated, fingers 2015secure chip 120 to the package.

[0150]FIG. 21 illustrates an alternative embodiment for attaching thechip to the package. A ridge 2110, having a notch 2115 at or near thetop of ridge 2110, encompasses the cavity 310. Chip 120 is wedged andheld into position by notch 2115. Thereafter, a process known as heatstaking is used to mount the chip. Heat staking includes applying heatand force at side 2111 of ridge, thus forcing ridge tightly against oraround chip 120.

[0151]FIG. 22 shows another embodiment of attaching a chip onto apackage. As shown, a channel 2250 surrounds cavity 310. A notch 2240 forreceiving the chip 120 is formed along or near the top of the cavity310. In some embodiments, a gasket or seal 2270 is placed at the bottomof the notch to ensure a tight seal when the chip is attached. Once thechip is located at the notch, a V-shaped wedge 2260 is inserted intochannel 2250. The wedge forces the body to press against chip's edgesand seal 2260, thus mating the chip to the package. This process isknown as compression sealing.

[0152] Other techniques such as insert molding, wave soldering, surfacediffusion, laser welding, shrink wrap, o-ring seal, surface etching, orheat staking from the top may also be employed.

[0153] C. Fluid Retention

[0154]FIG. 23 shows an alternative embodiment of package that employscheck valves to seal the inlets. As shown, depressions 2305 and 2315communicate with cavity 310 through inlets 350 and 360. Check valves2310 and 2320, which in some embodiments may be duck-billed checkvalves, are seated in depressions 2305 and 2315. To introduce a fluidinto the cavity, a needle is inserted into the check valve. When theneedle is removed, the check valve reseals itself to prevent leakage ofthe fluid.

[0155]FIG. 24 illustrates another package that uses reusable tape forsealing the cavity 310. As shown, a tape 2400 is located above inlets350 and 360. Preferably, end 2430 of tape is permanently fixed tosurface 2480 while end 2410 remains unattached. The mid section 2420 ofthe tape is comprised of non-permanent adhesive. This design allowsinlets to be conveniently sealed or unsealed without completelyseparating the tape from the package.

[0156]FIG. 25 illustrates yet another embodiment of the package thatuses lugs to retain fluids within the cavity. As shown, depressions 2520and 2530 communicate with cavity 310 via inlets 350 and 360. A plug2510, which in some embodiment may be composed of rubber or othersealing material, is mated to each of the depressions. Plugs 2510 areeasily inserted or removed for sealing and unsealing the cavity duringthe hybridization process.

[0157]FIG. 26a illustrates a package utilizing sliding seals forretaining fluids within the cavity. The seals are positioned in slots2610 that are located above the inlets. The slots act as runners forguiding the seals to and from the inlets. FIG. 26b illustrates the sealin greater detail. Seal 2640, which may be composed of rubber, teflonrubber, or other sealing material, is mated to each slot 2610. The sealincludes a handle 2650 which extends through the slot. Optionally, thebottom of the seal includes an annular protrusion 2645 to ensure matingwith inlet 350. The inlet is sealed or unsealed by positioning the sealappropriately along the slot. Alternatively, spring loaded balls, rotaryball valves, plug valves, or other fluid retention techniques may beemployed.

[0158] d. Chip Orientation

[0159]FIGS. 27a-27 b illustrate an alternative embodiment of thepackage. FIG. 27a illustrates a top view and FIG. 27b shows a crosssectional view. As shown, package 2700 includes a cavity 2710 on asurface 2705. A chip 2790 having an array of probes 2795 on surface 2791is mated to the bottom of cavity 2710 with an adhesive 2741. Theadhesive, for example, may be silicone, adhesive tape, or otheradhesive. Alternatively, clips or other mounting techniques may beemployed. Optionally, the bottom of the cavity may include a depressionin which a chip is seated.

[0160] This configuration provides several advantages such as: 1)permitting the use of any type of substrate (i.e., non-transparent ornon-translucent), 2) yielding more chips per wafer since the chip doesnot require an edge for mounting, and 3) allowing chips of various sizesor multiple chips to be mated to the package.

[0161] A cover 2770 is mated to the package for sealing the cavity.Preferably, cover 2770 is composed of a transparent or translucentmaterial such as glass, acrylic, or other material that is penetrable bylight. Cover 2270 may be mated to surface 2705 with an adhesive 2772,which in some embodiments may be silicone, adhesive film, or otheradhesive. Optionally, a depression may be formed around the cavity suchthat surface 2271 of the cover is at least flush with surface 2705.Alternatively, the cover may be mated to surface 2705 according to anyof the chip attachment techniques described herein.

[0162] Inlets 2750 and 2751 are provided and communicate with cavity710. Selected fluids are circulated through the cavity via inlets 2750and 2751. To seal the fluids in the cavity, a septum, plug, or otherseal may be employed. In alternative embodiments, any of the fluidretention techniques described herein may be utilized.

[0163] e. Parallel Hybridization and Diagnostics

[0164] In an alternative embodiment, the body is configured with aplurality of cavities. The cavities, for example, may be in a 96-wellmicro-titre format. In some embodiments, a chip is mounted individuallyto each cavity according to the methods described above. Alternatively,the probe arrays may be formed on the wafer in a format matching that ofthe cavities. Accordingly, separating the wafer is not necessary beforeattaching the probe arrays to the package. This format providessignificant increased throughput by enabling parallel testing of aplurality of samples.

[0165] V. Details of an Agitation System

[0166]FIG. 28 illustrates an agitation system in detail. As shown, theagitation system 2800 includes two liquid containers 2810 and 2820,which in the some embodiments are about 10 milliliters each. Container2810 communicates with port 350 via tube 2850 and container 2820communicates with port 360 via tube 2860. An inlet port 2812 and a ventport 2811 are located at or near the top of container 2810. Container2820 also includes an inlet port 2822 and a vent 2821 at or near itstop. Port 2812 of container 2810 and port 2822 of container 2820 areboth connected to a valve assembly 2828 via valves 2840 and 2841. Anagitator 2801, which may be a nitrogen gas (N2) or other gas, isconnected to valve assembly 2828 by fitting 2851. Valves 2840 and 2841regulate the flow of N₂ into their respective containers. In someembodiments, additional containers (not shown) may be provided, similarto container 2810, for introducing a buffer and/or other fluid into thecavity.

[0167] In operation, a fluid is placed into container 2810. The fluid,for example, may contain targets that are to be hybridized with probeson the chip. Container 2810 is sealed by closing port 2811 whilecontainer 2820 is vented by opening port 2821. Next, N₂ is injected intocontainer 2810, forcing the fluid through tube 2850, cavity 310, andfinally into container 2820. The bubbles formed by the N₂ agitate thefluid as it circulates through the system. When the amount of fluid incontainer 2810 nears empty, the system reverses the flow of the fluid byclosing valve 2840 and port 2821 and opening valve 2841 and port 2811.This cycle is repeated until the reaction between the probes and targetsis completed.

[0168] In some applications, foaming may occur when N₂ interacts withthe fluid. Foaming potentially inhibits the flow of the fluid throughthe system. To alleviate this problem, a detergent such as CTAB may beadded to the fluid. In one embodiment, the amount of CTAB added is about1 millimolar. Additionally, the CTAB affects the probes and targetspositively by increasing the rate at which they bind, thus decreasingthe reaction time required.

[0169] The system described in FIG. 28 may be operated in an alternativemanner. According to this technique, back pressure formed in the secondcontainer is used to reverse the flow of the solution. In operation, thefluid is placed in container 2810 and both ports 2811 and 2821 areclosed. As N₂ is injected into container 2810, the fluid is forcedthrough tube 2850, cavity 310, and finally into container 2820. Becausethe vent port in container 2820 is closed, the pressure therein beginsto build as the volume of fluid and N₂ increases. When the amount offluid in container 2810 nears empty, the flow of N₂ into container 2810is terminated by closing valve 2840. Next, the circulatory system isvented by opening port 2811 of container 2810. As a result, the pressurein container 2820 forces the solution back through the system towardcontainer 2810. In one embodiment, the system is injected with N₂ forabout 3 seconds and vented for about 3 seconds. This cycle is repeateduntil hybridization between the probes and targets is completed.

[0170]FIG. 29 illustrates an alternative embodiment of the agitationsystem. System 2900 includes a vortexer 2910 on which the chip package300 is mounted. A container 2930 for holding the fluid communicates withinlet 350 via tube 2950. A valve 2935 may be provided to control theflow of solution into the cavity. In some embodiments, circulator 2901,which may be a N₂ source or other gas source, is connected to container2930. Alternatively, a pump or other fluid transfer device may beemployed. The flow of N₂ into container 2930 is regulated by a valve2936. Circulator 2901 is also connected to inlet tube 2950 via a valve2902.

[0171] A waste container 2920 communicates with port 360 via outlet tube2955. In one embodiment, a liquid sensor 2940 may be provided forsensing the presence of liquid in outlet tube 2955. Access to the wastecontainer may be controlled by a valve 921. Optionally, additionalcontainers (not shown), similar to container 2930, may be employed forintroducing a buffer or other fluid into the cavity.

[0172] The system is initialized by closing all valves and fillingcontainer 2930 with, for example, a fluid containing targets. Next,valves 2936, 2935, and 2955 are opened. This allows N₂ to entercontainer 2930 which forces the fluid to flow through tube 2950 and intothe cavity. When the cavity is filled, valves 2935, 2936, and 2955 areclosed to seal the fluid in the cavity. Next, the vortexer is activatedto vibrate the chip package, similar to a paint mixer. In someembodiments, the vortexer may vibrate the package at about 3000 cyclesper minutes. The motion mixes the targets in the fluid, shortening theincubation period. In some embodiments, the vortexer rotates the chippackage until hybridization is completed. Upon completion, valve 2902and 2955 are opened to allow N₂ into the cavity. The N₂ empties thefluid into waste container 2920. Subsequently, the cavity may be filledwith a buffer or other fluid.

[0173]FIG. 30 illustrates an alternative embodiment in which theagitation system is partially integrated into the chip package. Asshown, chip package 300 includes a cavity 310 on which the chip ismounted. Cavity 310 is provided with inlets 360 and 350. The packagealso includes chambers 3010 and 3020. A port 3021 is provided in chamber3010 and is connected to inlet 360 by a channel 3025.

[0174] Chamber 3010 is equipped with ports 3011 and 3012. Port 3012communicates with inlet 350 through a channel 3015. Channel 3015 isprovided with a waste port 3016 that communicates with a fluid disposalsystem 3500 via a tube 3501. A valve 3502 regulates the flow of fluidsinto the disposal system. In some embodiments, the disposal systemincludes a waste container 3510 and fluid recovery container 3520 whichare connected to tube 3501. A valve 3530 is provided to direct the flowof fluids into either the waste container or recovery container.

[0175] Port 3011 is coupled to a fluid delivery system 3600 through atube 3601. Fluids flowing into chamber 3010 from the fluid deliverysystem are regulated by a valve 3602. The fluid delivery system includesfluid containers 3610 and 3620 that are interconnected with a tube 3690.Container 3610, which may hold a fluid containing targets, includesports 3616 and 3615. Port 3616 is connected to tube 3690. A valve 3612controls the flow of the fluid out of container 3610. A circulator 3605,which may be a N₂ source, is connected to port 3615 of container 3610.Alternatively, any type of gas, pump or other fluid transfer device maybe employed. The flow of N₂ into container 610 is controlled by a valve3618. A valve 3619 may also be provided to vent container 610.

[0176] Container 3620, which may hold a buffer, is provided with ports3625 and 3626. Circulator 3605 is connected to port 3625. A valve 3621is provided to control the flow of N₂ into container 3620. Port 3626 isconnected to tube 3690 via a valve 3622. Valve 3622 regulates the flowof the buffer out of container 3620. Optionally, additional containers(not shown), similar to container 3620, may be configured forintroducing other fluids into the cavity. A valve 3690 connectscirculator 3605 to tube 3690 for controlling the flow of N₂ directlyinto the package. A valve 3652 is provided for venting the fluiddelivery system.

[0177] In the initial operating state, all valves are shut. To start thehybridization process, a fluid containing targets is introduced intochamber 301 by opening valves 3602, 3612 and 3618. This injects N₂ intocontainer 3610 which forces the fluid to flow through 3601 and intochamber 3010. When chamber 3010 is filled, valves 3612 and 3618 areclosed. Next, valve 3642 is opened, allowing N₂ to flow directly intochamber 3010. The N₂ agitates and circulates the fluid into cavity 310and out to chamber 3020. As the volume of fluid and N₂ in chamber 3020increase, likewise does the pressure therein. When chamber 3020approaches its capacity, valve 3642 is closed to stop the fluid flow.Thereafter, the system is vented by opening valve 3652. Venting thesystem allows the back pressure in chamber 3020 to reverse the flow offluids back into chamber 3010. When chamber 3010 is filled, valve 3652is closed and valve 3642 is opened to reverse the fluid flow. This cycleis repeated until hybridization is completed.

[0178] When hybridization is completed, the system may be drained. Thisprocedure depends on which chamber the fluid is located in. If the fluidis located in chamber 3020, then valve 3502 is opened, while valve 3530is positioned to direct the fluid into the appropriate container(recovery or waste). The pressure in chamber 3020 forces the fluidthrough port 3016, tube 3501, and into the disposal system. If the fluidis in chamber 3010, then valve 3502 and 3642 are opened. As a result, N₂forces the fluid in chamber 3010 through port 3501 and into the disposalsystem.

[0179] Once the system is emptied, all valves are closed. A buffer orother fluid may be introduced into the cavity. For example, the cavitymay be filled with a buffer by opening valves 3601, 3621, and 3622. Thisinjects N₂ into container 3620 which forces the buffer therein to flowthrough the system until it fills cavity 310. In the alternative,ultrasonic radiation, heat, magnetic beads, or other agitationtechniques may be employed.

[0180] The present inventions provide commercially feasible devices forpackaging a probe chip. It is to be understood that the abovedescription is intended to be illustrative and not restrictive. Manyembodiments will be apparent to those skilled in the art upon reviewingthe above description. Merely as an example, the package may be moldedor machined from a single piece of material instead of two. Also, otherasymmetrical designs may be employed to orient the package onto thedetection systems.

[0181] The scope of the invention should, therefore, be determined notwith reference to the above description, but instead should bedetermined with reference to the appended claims along with their fullscope of equivalents.

What is claimed is:
 1. A method of making probe chips comprising thesteps of: forming a plurality of probe arrays on a substrate; separatingsaid substrate into a plurality of chips, each of said chips comprisingat least one probe array thereon; and mating at least one of said chipsto a package, said package comprising a reaction chamber, said reactionchamber comprising inlets for flowing fluid therein, said at least oneprobe array in fluid communication with said reaction chamber.
 2. Themethod as recited in claim 1 wherein said package is made by the stepsof: injection molding first and second halves of said package; andmating said first and second halves together.
 3. The method as recitedin claim 2 wherein one of said halves comprises flow channels therein,said flow channels in communication with said inlets.
 4. The method asrecited in claim 3 further comprising the step of applying a reenterableseal to flow channels in said package.
 5. The method as recited in claim1 wherein said substrate comprises alignment marks for forming saidprobe arrays thereon in a desired position, and wherein said alignmentmarks are used to identify locations for said separating of saidsubstrate into chips.
 6. The method as recited in claim 1 wherein saidpackage comprises an alignment structure thereon, wherein said step ofmating said chip to said package uses said alignment structures toposition said package at a desired position.
 7. The method as recited inclaim 1 wherein said package comprises an alignment structure thereon,and further comprising the step of identifying the location of at leastone target on said probe array in a scanner, wherein said package isplaced at a desired location in said scanner using said alignmentstructure.
 8. The method as recited in claim 1 wherein said step offorming a plurality of probe arrays comprises the steps of: selectivelyexposing said substrate to light; coupling selected monomers to saidsubstrate where said substrate has been exposed to light.
 9. The methodas recited in claim 1 wherein said step of separating comprises thesteps of: scribing said substrate in desired locations; breaking saidsubstrate along said scribe lines.
 10. The method as recited in claim 1wherein said step of forming a plurality of probe arrays on saidsubstrate is a step of forming a plurality of oligonucleotide probearrays on said substrate.
 11. The method as recited in claim 10 furthercomprising the steps of flowing labeled oligonucleotide target moleculesthrough said reaction chamber and identifying where said targetmolecules have bound to said substrate.
 12. The method as recited inclaim 11 wherein said package comprises a temperature probe and furthercomprising the step of monitoring and adjusting a temperature in saidreaction chamber.
 13. The method as recited in claim 1 wherein saidpackage is formed by the steps of: forming first and second packageportions; and acoustically welding said first and second packageportions together.
 14. The method as recited in claim 1 wherein saidstep of mating said chips to packages comprises the step of binding saidchips to said package with an adhesive.
 15. The method as recited inclaim 14 wherein said packages comprise a recessed region thereon,whereby said chips do not extend above a surface of said packages. 16.The method as recited in claim 1 further comprising the step of flowingtarget molecules through said reaction chamber.
 17. An apparatus forpackaging a substrate, said apparatus comprising: a substrate having afirst surface and a second surface, said first surface comprising aprobe array; a body having a mounting surface with a fluid cavity, saidsecond surface attached to said cavity; and a cover attached to saidmounting surface for sealing said cavity.
 18. The apparatus of claim 17wherein said cavity comprises an inlet port and an outlet port, saidinlet and outlet ports permitting fluids to circulate into and throughsaid cavity.
 19. The apparatus of claim 18 wherein said inlet and outletports comprise a reenterable seal.
 20. The apparatus of claim 17 whereinsaid probe array comprises an array of oligonucleotide probes.
 21. Anapparatus for packaging a substrate, said apparatus comprising: asubstrate having a first surface and a second surface, said firstsurface comprising a probe array and said second surface being an outerperiphery of said first surface; a body having a mounting surface, anupper surface, and a cavity bounded by said mounting surface and saidupper surface, said second surface being attached to said cavity andsaid first surface being within said cavity; and a cover attached tosaid mounting surface for defining an upper boundary to said cavity;wherein said cavity comprises a diffuser and a concentrator, saiddiffuser and said concentrator permitting laminar fluid flow throughsaid cavity.
 22. The apparatus of claim 21 wherein said probe arraycomprises an array of oligonucleotide probes.
 23. The apparatus of claim21 wherein said cover comprises a depression for receiving a temperaturecontrol element to maintain a reaction temperature in said cavity. 24.The apparatus of claim 21 wherein said cover comprises a first halfmated to a second half.
 25. The apparatus of claim 24 wherein said firsthalf comprises a first channel and a second channel, said first channelbeing in fluid communication with said diffuser and said second channelbeing in fluid communication with said concentrator.
 26. The apparatusof claim 25 wherein said second half comprises a third channel and afourth channel, said third channel being in fluid communication withsaid first channel, and said fourth channel being in fluid communicationwith said second channel.
 27. The apparatus of claim 26 wherein saidfirst channel and said second channel comprise re-enterable seals forsealing fluid in said cavity.